Silibinin attenuates ferroptosis in acute kidney injury by targeting FTH1

Abstract

Acute kidney injury (AKI) is primarily caused by renal ischemia-reperfusion injury (IRI), which is one of the most prevalent triggers. Currently, preventive and therapeutic measures remain limited. Ferroptosis plays a significant role in the pathophysiological process of IRI-induced AKI and is considered a key target for improving its outcomes. Silibinin, a polyphenolic flavonoid, possesses diverse pharmacological properties and is widely used as an effective therapeutic agent for liver diseases. Recent studies have reported that silibinin may improves kidney diseases, though the underlying mechanism remain unclear. In this study, we investigated whether silibinin protects against IRI-induced AKI and explored its mechanism of action. Our findings indicated that pretreatment with silibinin alleviated renal dysfunction, pathological damage, and inflammation in IRI-AKI mice. Furthermore, the results demonstrated that silibinin inhibited ferroptosis both in vivo and in vitro. Proteome microarrays were used to identify silibinin's target, and our results revealed that silibinin binds to FTH1. This binding affinity was confirmed through molecular docking, SPRi, CETSA, and DARTS. Additionally, co-IP assays demonstrated that silibinin disrupted the NCOA4-FTH1 interaction, inhibiting ferritinophagy. Finally, the inhibitory effects of silibinin on ferroptosis were reversed by knocking down FTH1 in vitro. In conclusion, our study shows that silibinin effectively alleviates AKI by targeting FTH1 to reduce ferroptosis, suggesting that silibinin could be developed as a potential therapeutic agent for managing and treating AKI.

Keywords: Acute kidney injury; Ferritin heavy chain 1; Ferritinophagy; Ferroptosis; Silibinin.

Graphical abstract

Fig. 1

Silibinin protects against renal dysfunction and pathological damage in IRI-AKI mice. (A) Chemical structure of silibinin (from PubChem Compound Database). (B) The schematic diagram of experimental design. (C–D) The BUN and Scr levels (n = 8). (E) Representative gross-morphological images of kidney cross section. (F, H) H&E and PAS staining of kidney sections, and renal injury score (n = 6, scale bar = 100 μm). (G, I-J) IHC sections and quantitative results of NGAL and Kim-1 staining (n = 6, scale bar = 100 μm). (K–L) Relative mRNA expression of NGAL and Kim-1 (n = 6). ^###P < 0.001 versus the sham group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the model group.

Fig. 2

Silibinin ameliorates renal macrophage infiltration and inflammation in IRI-AKI mice. (A–B) IHC sections and quantitative results of F4/80 staining (n = 6, scale bar = 100 μm). (C–E) The ELISA results of IL-1β, IL-6 and TNF-α of kidney homogenates (n = 8). (F–H) Relative mRNA expression of IL-1β, IL-6 and TNF-α (n = 6). ^##P < 0.01, ^###P < 0.001 versus the sham group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the model group.

Fig. 3

Silibinin inhibits ferroptosis in IRI-AKI mice. (A–C) GSH, MDA and SOD levels of kidney homogenates (n = 8). (D–E) TUNEL assay of kidney sections and TUNEL positive cell ratio (n = 5–6, scale bar = 100 μm). (F–H) Western bolt analysis and quantitative results of GPX4 and HO-1 (n = 3). (I–J) Relative mRNA expression of GPX4 and HO-1 (n = 6). (K) Ferrous iron levels of kidney homogenates (n = 8). (L–M) Prussian blue staining of kidney sections and quantitative results (n = 3, scale bar = 100 μm). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the sham group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the model group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Silibinin inhibits renal tubular cell ferroptosis induced by erastin. (A–B) MTT assay of HK-2 and NRK-52E cells. (C–H) GSH, MDA and SOD levels of HK-2 and NRK-52E cells. (I–J) Flow cytometry results of lipid ROS of HK-2 cells. (K–L) Flow cytometry results of lipid ROS of NRK-52E cells. (M–N) Ferrous iron levels of HK-2 and NRK-52E cells. (O–Q) Western bolt analysis and quantitative results of GPX4 and HO-1 of HK-2 and NRK-52E cells. (R–U) Relative mRNA expression of GPX4 and HO-1 of HK-2 and NRK-52E cells. (V–W) JC-1 assay and quantitative results of HK-2 cells (scale bar = 50 μm). (X–Y) JC-1 assay and quantitative results of NRK-52 cells (scale bar = 50 μm). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3.

Fig. 5

FTH1 plays a critical role in silibinin-mediated inhibition of ferroptosis, and silibinin inhibits ferroptosis by disrupting the NCOA4-FTH1 interaction. (A) The schematic diagram of identifying silibinin target proteins. (B) Venn diagram of silibinin target proteins and biotin target proteins. (C) Representative images of FTH1 in the proteome microarray. (D) Z-score and IMean ratio of FTH1-silibinin binding. (E) Molecular docking of FTH1 and silibinin. (F) SPRi fitting curves for silibinin to FTH1. (G–H) CETSA-Western blot analysis showed the protection of FTH1 by silibinin at different temperature gradients (n = 3). (I–J) DARTS-Western blot analysis showed the resistance of FTH1 to pronase digestion under the treatment of silibinin (n = 3). (K–M) Western bolt analysis and quantitative results of NCOA4 and FTH1 (n = 4). (N) Representative immunofluorescence images of FTH1 and NCOA4 co-staining. (O) Co-IP assay of NCOA4 and FTH1 interaction in the kidney of IRI mice. (P) Quantitative results of co-IP assay showing the endogenous interaction between NCOA4 and FTH1 (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 6

Knockdown of FTH1 alleviates the ferroptosis-inhibiting effect of silibinin. (A–B) Relative mRNA expression of FTH1 transfected with siRNA in 24h and 48h. (C) MTT assay of HK-2 cells transfected with FTH1 siRNA. (D–F) GSH, MDA and SOD levels of HK-2 cells. (G–H) Flow cytometry results of lipid ROS of HK-2 cells. (I) Ferrous iron levels of HK-2 cells. (J–K) Western bolt analysis and quantitative results of GPX4 of HK-2 cells. (L) Relative mRNA expression of GPX4. (M–N) JC-1 assay and quantitative results of HK-2 cells (scale bar = 50 μm). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3.